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@proceedings{1323999,
author = {Ilgová, Jana and Jedličková, Lucie and Dvořáková, Hana and Mikeš, Libor and Gelnar, Milan and Kašný, Martin},
booktitle = {4th Workshop of European Centre of IchthyoParasitology},
keywords = {Eudiplozoon nipponicum; cystatin; recombinant protein},
language = {eng},
isbn = {978-80-210-8016-4},
title = {Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea)},
year = {2015}
}
TY - CONF
ID - 1323999
AU - Ilgová, Jana - Jedličková, Lucie - Dvořáková, Hana - Mikeš, Libor - Gelnar, Milan - Kašný, Martin
PY - 2015
TI - Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea)
SN - 9788021080164
KW - Eudiplozoon nipponicum
KW - cystatin
KW - recombinant protein
N2 - Eudiplozoon nipponicum (Monogenea, Diplozooidae) typically inhabiting the gills of carp (Cyprinus carpio) is widely distributed Eurasian blood-feeding ectoparasite. The foregoing research of diplozoid Monogenea has been predominatly oriented towards understanding morphology and ecology of particular species. Nevertheless there is a lack of information regarding functional molecules synthesized by this taxonomic group. Among important molecules of various parasitic species belong cystatins (inhibitors of cystein peptidases). Besides regulation of endogenous processes in parasite bodies they play a substantial role e.g. in manipulation of the host immune system and/or regulation of blood digestion process. The aim of our experimental work is to reveal the presence, structure and function of E. nipponicum cystatin DNA/protein molecules using bioinformatic, molecular and proteomic methods. The gene coding cystatin of E. nipponicum was expressed in heterologous E. coli (BL21) system using pET19b expression vector. IPTG induced expression (22 °C, 6 hours) was analyzed on SDS-PAGE and soluble recombinant cystatin of E. nipponicum was purified on Ni-NTA agarose column. Production of cystatin was verified by both mass spectrometry (Orbitrap) and Western Blot using His-tag specific antibodies. Activity of recombinant inhibitor was tested by inhibitory assay using recombinant E. nipponicum cathepsin L and fluorogenic substrate Z-FR-AMC. Soluble recombinant cystatin was used for immunization of ICR mice. The polyclonal anti-cystatin sera reacted specifically with the crude extract of E. nipponicum and recombinant cystatin of E. nipponicum (Western blot).
ER -
ILGOVÁ, Jana, Lucie JEDLIČKOVÁ, Hana DVOŘÁKOVÁ, Libor MIKEŠ, Milan GELNAR a Martin KAŠNÝ. Cystatin: the cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea). In \textit{4th Workshop of European Centre of IchthyoParasitology}. 2015. ISBN~978-80-210-8016-4.
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